| Title |
Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35
|
|---|---|
| Published in |
Applied Microbiology and Biotechnology, May 2018
|
| DOI | 10.1007/s00253-018-9021-6 |
| Pubmed ID | |
| Authors |
Menglin Zhao, Jiaxian Wang, Manyu Luo, Han Luo, Meiqi Zhao, Lei Han, Mengxiao Zhang, Hui Yang, Yueqing Xie, Hua Jiang, Lei Feng, Huili Lu, Jianwei Zhu |
| Abstract |
Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines. |
X Demographics
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Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 4 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 4 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 100 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 100 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 21 | 21% |
| Student > Master | 15 | 15% |
| Researcher | 15 | 15% |
| Student > Doctoral Student | 6 | 6% |
| Student > Bachelor | 6 | 6% |
| Other | 6 | 6% |
| Unknown | 31 | 31% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 36 | 36% |
| Agricultural and Biological Sciences | 17 | 17% |
| Chemical Engineering | 3 | 3% |
| Engineering | 3 | 3% |
| Pharmacology, Toxicology and Pharmaceutical Science | 2 | 2% |
| Other | 8 | 8% |
| Unknown | 31 | 31% |
Attention Score in Context
This research output has an Altmetric Attention Score of 12. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 22 August 2023.
All research outputs
#2,865,218
of 24,387,992 outputs
Outputs from Applied Microbiology and Biotechnology
#412
of 8,090 outputs
Outputs of similar age
#57,578
of 334,365 outputs
Outputs of similar age from Applied Microbiology and Biotechnology
#6
of 144 outputs
Altmetric has tracked 24,387,992 research outputs across all sources so far. Compared to these this one has done well and is in the 88th percentile: it's in the top 25% of all research outputs ever tracked by Altmetric.
So far Altmetric has tracked 8,090 research outputs from this source. They receive a mean Attention Score of 4.3. This one has done particularly well, scoring higher than 94% of its peers.
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We're also able to compare this research output to 144 others from the same source and published within six weeks on either side of this one. This one has done particularly well, scoring higher than 96% of its contemporaries.